Hemoglobin F (fetal hemoglobin)

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TEST
Hemoglobin F (fetal hemoglobin)
METHOD
TEST EVALUATION
Sherry Woodhouse, M.D.
Fetal hemoglobin is more resistant to alkaline denaturation than other hemoglobins.
First, all hemoglobin species are converted to their corresponding
cyanomethemoglobin forms. A hemolysate is then exposed to alkali followed by
neutralization and precipitation of denatured adult hemoglobin with ammonium
sulfate. The absdrbance of the filtrate containing only the alkali-resistant fetal
hemoglobin is measured and compared to the absorbance of the total hemolysate.
Results are expressed as the fraction (%) of total hemoglobin that is resistant to
denaturation.
CLINICAL APPLICATIONS
Hemoglobin F (HbF) is the main red cell hemoglobin component in fetal
development. At birth, HbF makes up between 60% and 90% of red cell hemoglobin.
This usually decreases to the adult range of 0-2% at 6 months of age, but this varies
from individual to individual.
Elevated levels of HbF, usually between 2-5%, have been reported in some cases of
hereditary spherocytosis, leukemia, aplastic anemia, megaloblastic anemia, and
carcinoma metastatic to bone.
Approximately 50% of patients with 3-thalassemia trait will have HbF levels of
2-5%. In patients with homozygous g-thalassemia, HbF is almost always markedly
increased, usually reaching levels of 15-100%.
Homozygotes for HbS may have no fetal hemoglobin or levels as high as 20%.
Heterozygotes for HbS have normal levels of HbF.
When fetal hemoglobin reaches levels of 15% in patients with no other hematologic
disorder, heterozygosity for hereditary persistence of fetal hemoglobin (HPFH)
should be suspected. This is an asymptomatic disease found primarily in blacks and
Greeks. Although the findings in these two ethnic groups are similar, they are
thought to be of different genetic origins.
Homozygous HPFH is rare and found only in blacks. These people have 100% HbF
but no anemia. They may have mild hypochromia and microcytosis.
CLINICAL AND TECHNICAL LIMITATIONS
It is important to differentiate heterozygous HPFH from heterozygous
3-thalassemia. In HPFH, the sum of beta- and gamma-chain production is equal to
alpha-chain production. In thalassemia, there is a selective decrease in beta-chain

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synthesis and an associated hypochromia and mieroeytosis. HbF is evenly
distributed among all red cells in HPHF. In thalassemia, HbF is heterogeneouslv
distributed. This can be determined by a Kleihauer-Betke test. Hemoglobin F is
precipitated and fixed in red blood cells on a peripheral smear. Homogeneous versus
heterogeneous red cell populations can be determined.
The alkali denaturation method is fast and easy to perform. A significant
disadvantage is that HbF is not entirely resistant to alkaline denaturation and can be
underestimated by this method.
Another potential problem is that HbF may be absorbed by filter paper during
filtration. Choice of an appropriate grade of filter paper may minimize this
problem.
Other methods for measuring fetal hemoglobin include column chromatography,
radioimmunoassay, an enzyme-linked immunoabsorbent assay (ELISA) and radial
immunodiffusion.
REFERENCES
1. International Committee for Standardization in Haematology.
Recommendations for fetal hemoglobin reference preparations and fetal
haemoglobin determination by the alkali denaturation method. Br J Haematol
/&*• 42:133-136, 1979.
2. Molden DP, et al: Fetal hemoglobin: optimum conditions for its estimation by
alkali denaturation. Am J Clin Pathol 77:568-572, 1982.